Structural studies on induced antibodies with defined idiotypic specificities. II. The light chains of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

N-terminal amino acid sequence analyses have been performed on three preparations of light chains of A/J mice. Light chains derived from the IgG of unimmunized animals were compared to light chains of anti-p-azo-phenylarsonate (anti-Ar) antibodies possessing a cross-reacting idiotype (CRI); the latter were derived from the ascites fluid of a single A/J mouse, or from the pooled ascites fluids of 18 A/J mice. The heavy chains of these same two antibody preparations had previously been shown to comprise a single, homogeneous sequence to position 40. With few exceptions, the first 26 positions of light chains derived from unimmunized animals were extremely heterogeneous; the heterogeneity is comparable to that observed in a composite of sequence data on light chains of BALB/c myeloma proteins. Although the light chains obtained from anti-Ar antibodies possessing the CRI (whether from the pool of 18 A/J mice or from a single mouse) were more restricted in their sequence, at several positions as many as four alternative amino acids were detected. These studies indicate that an antibody population with defined idiotypic specificity, and very possibly identical heavy chain sequences, may contain at least four distinct light chains. The feasibility of structural studies on antibodies induced in individual mice is further demonstrated.     

Relation of a cross-reactive idiotype to genetic control of the immune response.

Antibodies elicited in strain A mice by immunization with keyhole limpet hemocyanin-p-azophenylarsonate (KLH-Ar) produce anti-Ar antibodies, some of which share a cross-reactive idiotype (CRI); in general, 20 to 70% of the antihapten antibody population carries the idiotype. Large amounts of antibody can be produced by the induction of ascitic fluids, using a 9:1 ratio of complete Freund's adjuvant (CFA) to antigen. Antibodies with the CRI can be isolated by isoelectric focusing from selected mice that have produced a high concentration of the CRI. The H chains exhibit a single homogeneous sequence through the first hypervariable region and, when isolated from a large number of individual mice, appear to be invariant in the first framework region. These findings indicate that somatic mutation is not a significant factor in the determination of framework sequences. Appearance of the CRI can be suppressed in adult A/J mice by administration of rabbit anti-idiotypic antiserum prior to immunization. Such suppressed mice produce normal concentrations of anti-Ar antibodies lacking the CRI. Anti-idiotypic antibodies produced against such antibodies failed to show cross-reactivity with anti-Ar antibodies arising in idiotypically suppressed or nonsuppressed A/J mice. The great sensitivity of the assay indicates that the number of such "private" idiotypes, all present on anti-Ar antibodies of a single strain, must be extremely large; this supports a somatic mechanism for the generation of diversity. The "private" idiotypes arising in suppressed, hyperimmunized mice can be adoptively transferred into multiple, irradiated (200 R) recipients by injections of spleen cells or of cells from ascitic fluids. The use of ascitic fluids permits the rapid production of a colony of mice bearing the idiotype. This should facilitate structural studies of a variety of idiotypically different molecules sharing the same (anti-Ar) specificity, as well as studies of the mechanism of suppression.     

Structural studies on induced antibodies with defined idiotypic specificities. V. The complete amino acid sequence of the light chain variable regions of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

The complete amino acid sequence of the variable regions of light chains derived from anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype is reported. At least two and probably more than three distinct light chains are associated with this idiotypically characterized antibody. The antibodies have several differences in their "framework" structures but evidence is presented indicating that all three light chain hypervariable regions have a homogeneous sequence. The data are discussed in relation to the various theories of antibody diversity. In addition, the findings support the view that hypervariable regions, idiotypic determinants, and the antibody-combining site involve, to a large extent, the same molecular structures.     

Comparison of the amino acid sequences of the variable domains of two homogeneous rabbit antibodies to type III pneumococcal polysaccharide.

The amino acid sequences of the V (variable) regions of the H (heavy) and L (light) chains derived from rabbit antibody K-25, specific for type III pneumococci, were determined; this is the second homogeneous rabbit antibody besides antibody BS-5 whose complete sequence of the V domain has been established (Jaton, 1974d). The V regions of L chains BS-5 and K-25 (both of allotype b4) differ from each other by 19 amino acid residues; 11 of these 19 substitutions are located within the three hypervariable sections of the V region. On the basis of seven amino acid differences within the N-terminal 28 positions, it is suggested that L chain K-25 belongs to a different subgroup of rabbit K chains and L chain BS-5. H chain K-25 (allotype a2) differs from another H chain of the same allotype by one amino acid substitution within the N-terminal 70 positions in addition to interchanges occurring in the first two hypervariable sections. H chain K-25 was compared with H chain BS-5 (allotype a1) and with the known V-region rabbit sequences. Allotype-related differences between a1, a2 and a3 chains appear to occur within the N-terminal 16 positions and possibly in scattered positions throughout the V-region. In the hypervariable positions, variability between the two antibodies is remarkably more pronounced within the third hypervariable section of both H and L chains than within the first two.     

Molecular mapping of idiotopes of anti-arsonate antibodies

As part of understanding molecular function in structural terms, we have been attempting to map the idiotypic topography of specific anti-arsonate (Ars) antibodies. A panel of anti-Ars hybridomas of which the complete primary sequences are known were used. These molecules show a varied reactivity profile with a panel of monoclonal anti-idiotypic antibodies. By judicious chain recombination experiments and chemical modifications that altered this reactivity profile, we were able to identify particular amino acid residues or discreet regions of anti-Ars antibodies as having crucial roles in the expression of idiotypic determinants. Idiotopes were mapped to the heavy chain second hypervariable region and D segment, and to the light chain first and third hypervariable regions.     

Idiotypic heterogeneity of the cross-reactive idiotype associated with the anti-p-azophenylarsonate antibodies of BALB/c mice

The cross-reactive idiotype (CRI) associated with the BALB/c antibody response against the p-azophenylarsonate (Ar) hapten was studied by analyzing hybridoma anti-Ar derived from BALB/c mice. The BALB/c CRI (CRIc) was previously thought to be a single idiotype as defined by rabbit anti-idiotype. CRIc has been resolved into at least two separate families of anti-Ar antibodies that are unrelated idiotypically to each other. Analysis of the 5AF6 family revealed considerable idiotypic heterogeneity, including a number of public idiotopes that appeared to be primarily associated with combinatorial idiotopes (requiring H + L chains) or L chain-specific idiotopes.     

Structural studies on induced antibodies with defined idiotypic specificities. VI. Amino terminal sequences of the heavy and light chain variable regions of anti-p-azophenylarsonate antibodies from A/J mice suppressed for a cross-reactive idiotype.

Previous studies in this series have been directed toward the elucidation of the heavy and light chain variable region structures of antibodies raised in A/J mice to the p-azophenylarsonate (Ar) hapten, certain of which bear a cross-reacting idiotype. The present study concerns an analysis of anti-Ar antibodies that arise in A/J mice suppresed for a cross-reacting idiotype. The results indicate that when an idiotype is suppressed and the animal subsequently hyperimmunized, the resultant antibodies are "deviated" into different V-region subgroups, both in the heavy and light polypeptide chains. The study presents the first primary structural analysis of a humoral immune response that has been manipulated by an idiotypic reagent.     

Structural evidence for a polymorphic or allelic form of the heavy chain variable region.

The heavy (H) chains of anti-phosphocholine (PC) antibodies from C57BL/6J and CBA/J were sequenced through the N-terminal 36 residues and compared with previously published sequences of A/J anti-PC antibody and BALB/c PC-binding myeloma proteins T15, M603, and M511. Each of these antibody preparations contained molecules having light (L) chains and idiotypic determinants of T15, M511, and M603 indicating the presence of at least three different anti-PC antibodies in each pool. The structures of the C57BL/6J and CBA/J H chains each revealed a single sequence from positions 1 to 36 (which includes the first complementarity determining region (CDR), and they were identical. The first CDR was identical to that previously found for BALB/c and A/J indicating that this portion of these antibody molecules is highly conserved throughout inbred mice and is probably critical to PC-binding. A surprising finding was that both C57NL/6 and CBA sequence differed from the BALB/c and A/J sequences at two positions, residue 14 and 16. Since each of these strains differs at the allotype locus, the data indicates that the evolution of allotypy in mice occurred after variable region diversity for the particular genes.     

A cross-reactive idiotype on anti-DNA antibodies defines a H chain determinant present almost exclusively on IgG antibodies

We have previously reported two anti-idiotypic antibodies, 3I and 8.12, that recognize L chain determinants on anti-DNA antibodies. We have generated a new anti-idiotypic antibody, F4, that recognizes a H chain determinant on cationic anti-DNA antibodies. F4 reactivity is present in high titer in serum of approximately 60% of SLE patients and on 84 of 706 myeloma proteins. It is preferentially associated with 3I reactive L chains. Furthermore, antibodies bearing both the F4 and 3I idiotypic determinants preferentially bind DNA. Amino acid sequencing of H chains isolated from four F4-reactive myeloma proteins suggests that they derive from two currently identified VH gene families. F4 reactivity is restricted almost exclusively to Ig of the IgG isotype suggesting that F4 may recognize either a somatically mutated hypervariable region or a variable region used late in the immune response. F4, therefore, represents a new idiotypic family preferentially associated with auto-Ag specificity and having features of an Ag-driven immune response.     

Characteristics of two idiotypic subfamilies of murine anti-p-azobenzenearsonate antibodies

A family of antibodies bearing a common or cross-reactive idiotype, termed CRIC, predominates in the response of most BALB/c mice to the p-azobenzenearsonate (Ar) hapten, but represents a minor component of the anti-Ar response of most A/J mice. Previous results have suggested that the VH region of CRIC is encoded by two different germ-line genes in both strains. We have determined extensive mRNA sequences for VH and VL, developed specific idiotypic reagents and measured affinities for two subfamilies of CRIC, designated CRIC1 and CRIC2. Both were found to be minor components of A/J anti-Ar antibodies, and CRIC1, but not CRIC2, is a major component of the BALB/c response. The two subfamilies utilize different VH germ-line genes but the same, or nearly identical V kappa genes. The VH nucleotide sequences of CRIC1 and CRIC2 exhibit approximately 90% homology. The D regions of both families are short (one or two amino acid residues) and some can be accounted for on the basis of known JH sequences alone. Affinity differences may account for the dominance of CRIA over CRIC1 and CRIC2 in A/J mice, but results obtained with allotype-congenic mice indicate that background (non-V region) genes are also important in controlling levels of expression of the CRIC1 idiotype. Our data suggest that the A/J germline VH gene that gives rise to the CRIC2 family of antibodies may be identical with a previously sequenced BALB/c germ-line VH gene. On the basis of these and earlier data it is suggested that extensive differences between inbred strains of mice in their complements of VH genes do not result from the accumulation of many mutations in these genes. An alternative possibility is that the differences arise from deletions and/or duplications of VH genes.     

The V-region sequence of the H chain from a third rabbit anti-pneumococcal antibody.

The amino acid sequence of the V (variable) region of the heavy (H) chain of rabbit antibody BS-1, raised against type III pneumococcal vaccine, is reported. Together with the sequence data of the V region of the light (L) chain previously determined [Jaton (1974a) Biochem. J. 141, 1-13], the present work completes the analysis of the V domain of the homogeneous antibody BS-1. The V domains (VL + VH regions) of this antibody are compared with those of two other anti-(type III) pneumococcal antibodies BS-5 and K-25 [Jaton (1975) Biochem. J. 147, 235-247]. Except for the second hypervariable section of the L chains, these antibodies have very different sequences in the hypervariable segments of the V domains. Within the third hypervariable region of the H chain, each antibody has a different length: BS-1 is three amino acids shorter than K-25 and two amino acids shorter than BS-5. When the sequences in that section are aligned for maximal homology, only two residues, glycine-97 and leucine-101, are common to the three antibodies. On the basis of the amino acid sequences of these three anti-pneumococcal antibodies, the results do not support the concept of a simple correlation between primary structure in the hypervariable sections (known to determine the shape of the combining site) and antigen-binding specificity.     

Molecular analysis of immunoglobulin heavy chain genes coding for idiotypic and anti-idiotypic antibodies involved in B-B cellular interaction.

We recently reported that a unique B cell clone (B19-1d), specific for a cross-reactive idiotype (CRI) on MOPC104E myeloma protein (M104E), enhances Igh-restricted CRI+ antibody production. In this paper, we report the nucleotide sequences of immunoglobulin heavy chain variable regions (VH) of both M104E and B19-1d-derived hybridoma (HB19) antibodies. The sequence data revealed that both belong to the J558 germ line VH gene subfamily. Strikingly, not only the VH region, but also the leader sequences of M104E and HB19 are very similar to each other at 88% (VH) and 91% (leader) homology, but they use different D and J segments. The VH region sequence similarity is highest among the germ line VH gene sequences of the BALB/c J558 subfamily so far screened. Southern hybridization data, using 5'-noncoding regions of either M104E or HB19 genomic VH gene clones as probes, revealed that both VH genes are conserved in the M104E CRI producer strains of mice. Moreover, these probes show the restriction length polymorphism pattern of mouse VH genes in various strains. That the HB19 VH gene locates to the 5' upper arm of the M104E VH gene on the chromosome was suggested by Southern blot hybridization. Immunoglobulin VH gene restriction of idiotypic and antiidiotypic B-B cellular interaction is discussed from a molecular point of view.     

The VK gene expressed by BALB/c ABPC48 cross-reactive idiotypes induced by anti-idiotypic immunization is identical to that of BALB/c anti-oxazolone and A/J anti-arsonate antibodies

Anti-idiotypic immunization triggers the production of antibodies that are structurally related to the idiotype. We have shown that the heavy chain variable regions of antibodies produced after anti-ABPC48 (A48) anti-idiotypic immunization of BALB/c mice are homologous to that of A48, except for the third hypervariable region. We present here partial light chain sequences of A48 and of antibodies induced by anti-idiotypic immunization. Nearly perfect homology is found, suggesting that these chains are the products of genes derived from a unique VK germ-line gene. These observations indicate that the H and L hypervariable regions contribute to define the structure of A48 idiotopes. Remarkably, the VK sequence we identify is the same as that described for anti-arsonate and anti-oxazolone antibodies. We discuss the relative importance of particular amino acids for idiotype expression and antigen-binding activity.     

Two induced anti-Z-DNA monoclonal antibodies use VH gene segments related to those of anti-DNA autoantibodies

The cDNA for H and L chain V regions of two anti-Z-DNA mAb, Z22 and Z44, were cloned and sequenced. These are the first experimentally induced anti-nucleic acid antibody sequences available for comparison with autoantibody sequences. Z22 and Z44 are IgG2b and IgG2a antibodies from C57BL/6 mice. They recognize different facets of the Z-DNA structure. They both use VH10 family genes and share 95% sequence base sequence identity in the VH and leader sequences; however, they differ in the 5'-untranslated region of the VH mRNA, indicating they arise from different germline genes. Both use JH4 segments. They differ from each other very extensively in the CDR3 of both H and L chains. The most closely related H chains in the current GenBank/EMBL data base are two mouse IgG anti-DNA autoantibodies, one from an MRL-lpr/lpr mouse (MRL-DNA4) and one from an NZB/NZW mouse (BV04-01). Z22 and Z44 share 95% sequence identity with these antibodies in the VH segment. In addition, Z22 is identical to MRL-DNA4 at 91% of the positions in the 5'-untranslated region of the H chain mRNA. The two antibodies share 95% base sequence identity in the V kappa segment. The most closely related L chains, with 97 to 98% sequence identity, are the V kappa 10b germline gene for Z22 and the V kappa 10a germ line gene, which is associated with A/J anti-arsonate antibodies and BALB/c anti-ABO blood group substance antibodies, for Z44. Z22 and Z44 share several structural features (similarities in VH, JH, and V kappa) but differ very markedly in the L chain CDR1 and both H and L chain CDR3 sequences; these regions may determine the differences in their specific interactions with Z-DNA.     

Purification, specificity, and hypervariable region sequence of anti-pneumococcal polysaccharide antibodies elicited in a single rabbit.

Four homogeneous antibodies to type VIII pneumococcal polysaccharide (S8) were isolated from the serum of a single rabbit (3322) by affinity chromatography on an S8 immunoadsoebent by utilizing gradient elution with cellobiose and NaCl. The binding properties of these antibodies were determined by a radioimmunoassay with 125I-bovine gamma-globulin-S8. Cellobiose (a disaccharide unit of S8) was the immunodominant group of each of the four antibodies, but each antibody bound to this disaccharide with different relative affinities. The amino acid sequences (positions 0-40) of three of the four antibody light chains were each different both in framework and first hypervariable region sequences. The fourth antibody light chain has a blocked amino terminus. These findings indicate that antibodies elicited by a relatively simple antigen and examined at one time during the course of immunization in a single rabbit may exhibit common specificities for an oligosaccharide determinant, yet have different binding affinities for that determinant as well as different primary structures in the complementarity (hypervariable) regions and framework regions.     

Immunoglobulin idiotype and anti-anti-idiotype utilize the same variable region genes irrespective of antigen specificity

Idiotypic determinants characterizing certain antibody specificities have been proven valuable structural and genetic markers in studies of antibody diversity and regulation. The heritable predominant idiotype associated with the response to p-azophenylarsonate in A/J mice consists of a set of highly homologous (greater than 95%) heavy and light chain variable region amino acid sequences probably arising by somatic mutation from one or a few V region genes. We examined a peculiar set of monoclonal antibodies that have been defined as CRI by serologic analysis, but that have no affinity for the hapten Ars. These antibodies were elicited by immunization with anti-CRI rather than by the conventional immunization with antigen. The amino acid sequences of the amino terminal half of the V regions of these anti-(anti-CRI) antibodies are indistinguishable from those of conventional Ars-binding CRI antibodies. Thus, Ars-binding CRI and Ars-nonbinding anti-(anti-CRI) are derived from similar or identical VH and VL genes.     

Towards a chemical definition of idiotypy.

The idiotypic determinants have been precisely located to the variable regions of immunoglobulin polypeptide chains. Both chains are generally required to express the idiotype. The major idiotypic determinants are the result of the amino acid sequence of the hypervaiable regions, although some idiotypic determinants reside outside the antibody combining site and these so called "framework idiotypes" are important markers. In my view the hypervariable regions are spatially disposed so as to present adequate antigenic stimulation, and they display enough structural heterogeneity to account for the uniqueness of the idiotype in the general population of immunoglobulin molecules. Hypervariable regions, the antibody combining site, and the idiotypic determinants thus amalgamate three formerly diverse concepts into a unified theoretical construct.     

Nucleotide sequence of messenger RNA encoding VHDJH and VKJK of a highly conserved idiotype-defined primary response anti-hapten antibody.

The primary humoral immune response of mice to the hapten phthalate (Xmp) is focused upon two adjacent immunodominant negatively charged carboxyl groups on a benzene ring that are in positions meta and para to the azolinkage (i.e., Xmp) to the protein carrier keyhole limpet hemocyanin. A significant fraction of the anti-Xmp antibodies raised in several different inbred mouse strains (BALB/c, DBA/2, A/HeHa; C3H, and SM/J), and many wild mouse populations express a cross-reactive Id, CRIXmp-1. This CRIXmp-1 is conspicuously absent in C57BL/6 mice. In order to obtain a better understanding of the events and parameters that influence the selection and regulation of the primary response B cell repertoire, and to explore the structural basis of Ag binding, we have determined the nucleotide sequence of the entire V region gene complexes, which encode the H and L chains of these highly conserved and dominant CRIXmp-1+ antibodies. Our data establish that the H chain gene complex consists of a single VH germ-line gene that is identical to VH Oxazolone-1, encoding the H chain of another highly conserved and dominant cross-reactive Id family associated with the primary response to Oxazolone. In CRIXmp-1+ Xmp-specific hybridomas this gene is joined to a limited set of D region sequences that express a conserved amino acid motif-GLR. At least three of the five D regions examined are coded for by DFL16.2. This VHD complex can be utilized with one of three different JH region genes (JH1, JH2, and JH4) without any significant effect upon antibody fine specificity or Id. In spite of this lack of JH fidelity all of the CRIXmp-1+ hybridomas have precisely maintained the same length in the H chain CDR3 and FRW4 by altering either the length of the D segment or the length of JH. Nucleotide sequence analysis of the VL gene complex of CRIXmp-1+ anti-Xmp antibodies indicates that the L chain V region is also encoded by a single germ-line gene. The amino acid sequence predicted from the nucleotide sequence of the VKJK from Xmp-specific CRIXmp-1+ hybridomas is identical to the sequence of the anti-arsonate antibody 1210.7, which is the prototype of another Id family (CRI) that is conserved and dominant in BALB/c mice.     

Analysis of antibody diversity: V-D-J mRNA nucleotide sequence of four anti-GAT monoclonal antibodies. A paucigene system using alternate D-J recombinations to generate functionally similar hypervariable regions

The nucleotide sequence of four anti-(Glu60-Ala30-Tyr10)n (GAT) monoclonal gamma 1 heavy chain mRNAs was determined from codon 10 to 120. This sequence overlaps with the NH2-terminal amino acid sequence, allowing elucidation of the complete protein sequence encompassing regions VH, D and JH. These sequences, which are highly conserved, indicate that anti-GAT antibodies expressing the same public idiotypic specificities represent a paucigene system, which uses at least two D-J combinations leading to functionally similar hypervariable regions involved in the recognition of the dominant Glu-Tyr determinant. D regions are encoded by D genes which are closely related either to the D-SP2 or the D.FL16 germ line gene cores.     

Structural and antigenic studies of an idiotype-bearing murine antibody to the arsonate hapten.

Mice of strain A/J responded to repeated intraperitoneal injection of Limulus hemocyanin derivatized with arsanilic acid by producing large quantities (approximately 5 mg/mL of ascites fluid) of IgG antibodies specific for this hapten. The antibodies possessed a characteristic idiotypic determinant and exhibited restricted heterogeneity as demonstrated by isoelectric focusing and primary N-terminal amino acid sequence analysis of isolated light and heavy polypeptide chains. Both light- and heavy-chain sequences were comparable to those of myeloma proteins in lack of heterogeneity. The N terminus of the light chain exhibited V KI sequence and only one position in the first 30 residues showed more than one amino acid. No variability was observed in the first 10 N-terminal residues of the heavy chain. Rabbit antiserum to the idiotype blocked binding of hapten by the purified antibody. The presence of both light- and heavy-chain antigenic determinants was required for optimal formation of the idiotypic determinant.